Lec9/10 - Enzymes

Question 1

What role can enzyme active site residues have besides catalytic activity?

Answer 1

Binding energy (non-covalent interactions)

Question 2

In what ways can enzyme active site residues be involved in catalysis?

Answer 2

Proton Donors/Acceptors (charged side chains); Groups that can form covalent bonds with substrates (e.g., nucleophiles, electrophiles) and Metal (co-factor) coordination

Question 3

State the Michaelis-Menten equation

Answer 3

V0 = Vmax[S] / Km + [S]

Question 4

How is Km defined and what do lower/higher Km values indicate?

Answer 4

Km is a measure of the affinity of an enzyme for a particular substrate -> lower Km = tighter binding

Question 5

How is Vmax defined?

Answer 5

Vmax is the initial velocity at which an enzyme is Saturated with substrate

Question 6

What is Kcat and what does it measure?

Answer 6

It is the catalytic constant/turnover number (it measures the number of molecules of substrate converted to product per active site per second)

Question 7

What is the specificity constant, and is lower or higher better?

Answer 7

Kcat / Km -> higher = “better” enzyme reaction (used to compare catalytic efficiency of different enzymes and relative abilities of different compounds to serve as a substrate for a particular enzyme)

Question 8

Name the 6 Classes of Enzymes

Answer 8

1. Oxidoreductases/Dehydrogenases
2. Transferases
3. Hydrolyses
4. Lyases
5. Isomerases
6. Ligases/Synthases

Question 9

What do Oxidoreductases do?

Answer 9

Catalyse redox reactions

Question 10

What do transferases do?

Answer 10

Catalyse group-transfer reactions

Question 11

What do hydrolyses do?

Answer 11

Catalyse hydrolysis (cleave a bond, with addition of water to products)

Question 12

What do lyases do?

Answer 12

Catalyse lysis (cleavage of C-C, C-O or C-N, leaving a double bond)

Question 13

What do isomerases do?

Answer 13

Catalyse structural change within a molecule without changing its molecular formula

Question 14

What do ligases do?

Answer 14

Catalyse ligation of two substrates, forming a new covalent bond (requires ATP)

Question 15

What feature of enzyme kinetics can suggest that an enzyme is regulated?

Answer 15

A sigmoidal v0 vs [S] plot (instead of hyperbolic) - this shows non-Michaelis-Menten kinetics

Question 16

What is meant by an Allosteric enzyme?

Answer 16

They undergo conformational change, brought about by the binding of small molecules to clefts in the protein surface

Question 17

What is meant by co-operativity between subunits?

Answer 17

The binding of a substrate to ONE active site affects SUBSEQUENT binding to all other subunits (usually more active form)

Question 18

Name the two forms of an allosteric enzyme, and state how binding of certain molecules stabilises one or the other.

Answer 18

Binding of an activator stabilises the active form (R-form)
Binding of an inhibitor stabilises the inactive form (T-form)

Question 19

Name the two molecules that regulate the PFK-1 allosteric enzyme (and what is significant about the inhibitor molecule)

Answer 19

ADP = activator; PEP = inhibitor (PEP is a product of a later reaction in the same pathway -> negative feedback for committed step of glycolysis)

Question 20

How do ADP and PEP affect the apparent Km of PFK-1

Answer 20

ADP (activator) -> lower apparent Km
PEP (inhibitor) -> higher apparent Km

Question 21

Define Competitive Inhibitors

Answer 21

Bind only to the enzyme (instead of the substrate), does not bind to ES complex

Question 22

Define Uncompetitive Inhibitors

Answer 22

Bind exclusively to the Enzyme-Substrate Complex, reducing product formation (does not bind to enzyme)

Question 23

Define Non-Competitive Inhibitors

Answer 23

Bind equally well to Enzyme or ES Complex, affecting the catalysis (ES -> E + P) step

Question 24

Describe the effects of competitive, uncompetitive and non-competitive inhibitors on Km and Vmax

Answer 24

Competitive: Increase Km
Uncompetitive: Decrease Vmax and Km
Non-Competitive: Decrease Vmax

Question 25

Describe the effects of competitive, uncompetitive and non-competitive inhibitors on the Lineweaver-Burk plot of 1/v0 vs 1/[s]

Answer 25

Competitive: steeper gradient, same y intercept (same 1/Vmax)
Uncompetitive: same gradient, greater y intercept (higher 1/Vmax)
Non-Competitive: steeper gradient, same x intercept (same 1-/Km)

Question 26

What types of enzyme inhibition are common in reality?

Answer 26

Competitive, and Mixed (more complex, but affects Vmax and ALSO Km)

Question 27

How is stereoisomerism related to enzymatic activity?

Answer 27

Enzymes can usually only act on one stereoisomer of a substrate

Question 28

What is significant about the shape of the transition state in an enzyme-catalysed reaction?

Answer 28

The active site is most complementary in shape to (and binds most tightly to) the transition state -> transition state STABILISATION lowers energy barrier

Question 29

How do enzymes increase the “effective” concentration of substrate?

Answer 29

Proximity effect: enzyme brings reacting substrates closer together